Redox regulation of cell functions by alpha-lipoate: biochemical and molecular aspects.
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Animals↗
Biomedical subjects
S Roy
Publications and source records attributed to S Roy.
A cognate tRNA specific conformational change in glutaminyl-tRNA synthetase and its implication for specificity.
Conformational changes that occur upon substrate binding are known to play crucial roles in the recognition and specific aminoacylation of cognate tRNA by glutaminyl-tRNA synthetase. In a previous study we had shown that glutaminyl-tRNA synthetase labeled selectively in a nonessential sulfhydryl residue by an environment sensitive probe, acrylodan, monitors many of the conformational changes that occur upon substrate binding. In this article we have shown that the conformational change that occurs upon tRNA(Gln) binding to glnRS/ATP complex is absent in a noncognate tRNA tRNA(Glu)-glnRS/ATP complex. CD spectroscopy indicates that this cognate tRNA(Gln)-induced conformational change may involve only a small change in secondary structure. The Van't Hoff plot of cognate and noncognate tRNA binding in the presence of ATP is similar, suggesting similar modes of interaction. It was concluded that the cognate tRNA induces a local conformational change in the synthetase that may be one of the critical elements that causes enhanced aminoacylation of the cognate tRNA over the noncognate ones.
2-Naphthylamine↗
The central response to ovarian carcinoma simulates the response to sepsis.
BACKGROUND: Animal models of stress and sepsis demonstrate increased hypophyseal gene expression of the transcription factor c-fos and the cytokines interleukin-1 and interleukin-6. Chronic central nervous system exposure to interleukin-1 results in hypermetabolism, accelerated nitrogen loss, anorexia, and cachexia. We test the hypothesis that the host response to ovarian carcinoma recapitulates the host response to sepsis regarding the elaboration of the transcription factors and cytokines in the central nervous system, liver, and lung. MATERIALS AND METHODS: Nude mice were seeded intraperitoneally with either ovarian carcinoma (MA-148) or vehicle. The animal subjects were observed for 5 weeks and sacrificed for brain, pituitary, lung, and liver mRNA. We studied the mRNA accumulation of the transcription factors c-fos, c-jun, and C/EBP alpha and the cytokines interleukin-1 and interleukin-6 using reverse-transcriptase polymerase chain reaction. RESULTS: Compared with the control, ovarian carcinoma in the mouse model resulted in the following: (1) Pituitary c-fos and c-jun mRNA increased 3-fold (P = 0.012) and 6-fold (P < 0.001), respectively; (2) pituitary IL-1 and IL-6 mRNA increased 4-fold (P < 0.001) and 8-fold (P = 0.037), respectively; (3) liver c-fos mRNA increased > 8-fold (P < 0.001); and (4) lung C/EBP alpha mRNA decreased greater than 10-fold (P < 0.001). CONCLUSIONS: We conclude that the host response to ovarian carcinoma in this animal model recapitulates many aspects of the host response to bacterial sepsis especially concerning pituitary gene expression. These data suggest that, as in sepsis, a hypothalamic-hypophyseal-mediated cytokine response in ovarian carcinoma may result in hypermetabolism, accelerated nitrogen loss, anorexia, and cachexia.
Animals↗
Use of a coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from Escherichia coli.
We have designed a novel coupled transcriptional construct wherein Escherichia coli uracil DNA glycosylase (UDG) and Bacillus subtilis phage PBS-2 encoded uracil DNA glycosylase inhibitor protein (Ugi) genes were cloned in tandem, downstream of an inducible promoter (Ptrc). Use of this bicistronic operon has allowed purification of large amounts of UDG-Ugi complex formed in vivo. The system has also been exploited for purification of large amounts of Ugi. While establishing the expression system, one of the constructs showed detectable suppression of UAG termination codon and resulted in accumulation of a minor population of a putative readthrough polypeptide corresponding to UDG. We discuss the likely occurrence of such a phenomenon in overproduction of other recombinant proteins. Finally, the usefulness of the operon construct in convenient mutational analysis to study the mechanism of UDG-Ugi interaction is also discussed.
Bacillus Phages↗
Morphine accelerates the progression of sepsis in an experimental sepsis model.
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Animals↗
Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube.
Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (beta-D-glucosyl)3 Yariv phenyl-glycoside [(beta-D-Glc)3] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(beta-D-Glc)3 complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall.
Cell Wall↗
Comparable renal graft survival in African-American and Caucasian recipients.
In past years, many pediatric transplant centers found African-American renal transplant recipients to have poor graft survival. Since 1991 anti-lymphocyte induction therapy has been routinely used for pediatric cadaveric (CAD) and living-related donor (LRD) renal allograft recipients at the University of Tennessee, Memphis. Sixteen African-American first renal allograft recipients received induction therapy: 11 CAD allografts (10 OKT3, 1 ATGAM) and five LRD (all ATGAM). Sixteen Caucasian recipients received induction therapy; 3 CAD (all OKT3), 1 living-unrelated donor (OKT3), and 12 LRD (9 ATGAM, 3 OKT3). Mean age at renal transplantation was 11.8 and 10.5 years for African-American and Caucasian recipients, respectively. Predicted graft survival (PGS) estimated by the Kaplan-Meier method for the African-American patients was 94% at both 1 and 3 years, and for Caucasian patients was 94% and 85% at 1 and 3 years, respectively. Eleven African-American CAD recipients had a PGS of 91% at 1 and 3 years. Renal allograft survival for African-American and Caucasian pediatric recipients at our center appears to be comparable. This could be due, in part, to the use of anti-lymphocyte induction therapy. However, other factors, such as improved compliance or better immunological and pharmacological monitoring, may also have contributed.
Adolescent↗
Abnormalities detected on transvaginal ultrasonography in tamoxifen-treated postmenopausal breast cancer patients may represent endometrial cystic atrophy.
OBJECTIVE: This study was conducted to examine the histopathologic changes in tamoxifen-treated postmenopausal patients with endometrial thickness > or = 5 mm with transvaginal ultrasonography. STUDY DESIGN: Thirty-five tamoxifen-treated postmenopausal breast cancer patients underwent transvaginal pelvic ultrasonography with endometrial thickness > or = 5 mm followed by either curettage-hysteroscopy (n = 24), or hysterectomy (n = 11). Endometrial histopathologic findings were examined. RESULTS: Overall, endometrial polyps were the most common histopathologic finding (23 of 35 patients). Endometrial cystic atrophy was uncommonly detected in patients undergoing curettage-hysteroscopy (1 of 24 patients) compared with patients undergoing hysterectomy (9 of 11 patients). No cases of endometrial cancer or hyperplasia were detected. CONCLUSIONS: Endometrial polyps were a frequent finding in tamoxifen-treated postmenopausal women who had endometrial thickness > or = 5 mm with the use of transvaginal ultrasonography. Endometrial cystic atrophy may explain "thickened endometrium" on transvaginal ultrasonography in this patient population with no evidence of endometrial polyps, hyperplasia, or adenocarcinoma after surgical evaluation.
Adult↗
Oral trovafloxacin compared with intravenous cefoxitin in the prevention of bacterial infection after elective vaginal or abdominal hysterectomy for nonmalignant disease. Trovafloxacin Surgical Group.
BACKGROUND: Trovafloxacin is a new fourth-generation fluoroquinolone whose pharmacokinetics and in vitro activity suggest that it is well suited for antibiotic prophylaxis in elective hysterectomy. METHODS: In a randomized, double-blind, multicenter study, parallel groups of women 18 years of age or older received either 200 mg trovafloxacin by mouth and intravenous (i.v.) placebo or 2 g cefoxitin by i.v. infusion and placebo by mouth before elective vaginal or abdominal hysterectomy for nonmalignant disease. RESULTS: In the 103 and 97 patients in the trovafloxacin and cefoxitin groups, respectively, who were evaluable for efficacy, the prophylactic success rates at hospital discharge (96% in both groups) and 30 +/- 6 days after hysterectomy (88% and 91% in the trovafloxacin and cefoxitin groups, respectively) were statistically equivalent. Both antibiotics were well tolerated. CONCLUSION: A single oral 200 mg dose of trovafloxacin is as effective and safe as a standard cefoxitin parenteral regimen in the prevention of primary bacterial infection after elective vaginal or abdominal hysterectomy for nonmalignant disease.
Administration, Oral↗
Treatment of acute gynecologic infections with trovafloxacin. Trovafloxacin Surgical Group.
BACKGROUND: Trovafloxacin, a broad-spectrum fourth-generation quinolone with gram-positive and gram-negative aerobic and anaerobic bacterial activity, is available in oral and intravenous formulations. The objective of this prospective, multicenter, double-blind, randomized study was to compare the efficacy of trovafloxacin with that of cefoxitin, an approved drug for treatment of acute gynecologic infections, together with amoxicillin/clavulanic acid as oral follow-on treatment. METHODS: Patients with a clinical diagnosis of acute pelvic infection received either intravenous alatrofloxacin with oral trovafloxacin follow-on (trovafloxacin) or a combined regimen of cefoxitin followed by amoxicillin/clavulanic acid for a maximum of 14 days. The primary endpoint was clinical response to therapy on follow-up at day 30. RESULTS: Clinical success rates were comparable between the trovafloxacin (n = 107) and comparative (n = 119) groups at study end (90% vs. 86%, respectively; 95% confidence interval, -4.5, 12.5). Among clinically evaluable patients, clinical success rates for infections involving Enterococcus species were higher with trovafloxacin than with the comparative regimen at the end of treatment (96% and 85%) and at study end (96% and 86%). CONCLUSION: Intravenous alatrofloxacin followed by oral trovafloxacin for a maximum of 14 days of total therapy was efficacious in the treatment of acute pelvic infections.
Acute Disease↗
mu-Opioid receptor-knockout mice: the role of mu-opioid receptor in gastrointestinal transit.
The role of mu-opioid receptor in gastrointestinal transit was investigated using mu-opioid receptor knockout mice (MOR-KO). Our result establishes unequivocally that inhibition of GI transit by morphine is a mu-opioid receptor mediated function. In addition, we show that neither delta nor kappa receptor agonist given supraspinally or peripherally are able to inhibit GI transit in MOR-KO animals. It was interesting to observe that basal GI motility was lower in MOR-KO (-/-) compared to heterozygous (+/-) and wild type (+/+) animals.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
Binding of peptides in solution by the Escherichia coli chaperone PapD as revealed using an inhibition ELISA and NMR spectroscopy.
PapD is the prototype member of a family of periplasmic chaperones which are required for assembly of virulence associated pili in pathogenic, gram-negative bacteria. In the present investigation, an ELISA has been developed for evaluation of compounds as inhibitors of PapD. Synthetic peptides, including an octamer, derived from the C-terminus of the pilus adhesin PapG were able to inhibit PapD in the ELISA. Evaluation of a panel of octapeptides in the ELISA, in combination with NMR studies, showed that the peptides were bound as extended beta-strands by PapD in aqueous solution. The PapD-peptide complex was stabilized by backbone to backbone hydrogen bonds and interactions involving three hydrophobic peptide side chains. This structural information, together with previous crystal structure data, provides a starting point in efforts to design and synthesize compounds which bind to chaperones and interfere with pilus assembly in pathogenic bacteria.
Amino Acid Sequence↗
Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis.
Caspase-mediated proteolysis of downstream substrates is a critical element of the execution pathway common to all forms of apoptosis studied to date. While this caspase-dependent pathway is activated during cytotoxic lymphocyte granule-induced cell death, recent studies have also provided evidence for caspase-independent pathways. However, the mechanisms mediating these additional pathways have not been defined. The current study demonstrates that DNA-PKcs and NuMA are directly and efficiently cleaved by granzyme B in vitro and in vivo, generating unique substrate fragments not observed during other forms of apoptosis. This direct, caspase-independent ability of granzyme B to cleave downstream death substrates constitutes an apoptotic effector mechanism that is insensitive to inhibitors of the signaling or execution components of the endogenous apoptotic cascade.
Antigens, Nuclear↗
In vitro function of an adjustable temporary venous spring filter: comparison with the temporary RF02 filter and the permanent Greenfield vena cava filter.
RATIONALE AND OBJECTIVES: The authors compared in vitro function of a temporary venous spring filter with that of a temporary RF02 filter and a permanent Greenfield filter. MATERIALS AND METHODS: All three types of filters were placed in thin polyethylene tubes (diameters, 10.0-18.0 mm). Physiologic saline was substituted for flowing blood, and blood clots of three sizes (6 x 10 mm, 6 x 20 mm, 9 x 20 mm) were funneled to the filters. Clot-trapping ability of each filter and elevation of intraluminal pressure after clot trapping were assessed for each tube size. RESULTS: No statistically significant elevation in intraluminal pressure was detected immediately after placement of any filter. The clot-trapping ability of the spring filter and of Greenfield filter were slightly lower than that of the RF02 filter, but the differences were not statistically significant. After filters had trapped large clots, a high pressure gradient was detected in the 10.0-mm tube for all filters. The spring filter was associated with a higher pressure than the other filters in the 12.0-mm tube (P < .05). CONCLUSION: In vitro function of the spring filter was satisfactory in comparison with that of the RF02 filter and the Greenfield filter. For efficient filtering in the inferior vena cava, development of a larger version of the filter may be necessary.
Equipment Design↗
Determination of absorption characteristics of AG337, a novel thymidylate synthase inhibitor, using a perfused rat intestinal model.
The purpose of this study was to determine the intestinal absorption characteristics of AG337, a mechanism-based inhibitor of thymidylate synthase, using a perfused rat intestinal model. Effects of site, pH, temperature, concentration, Na+, and inhibitors on the absorption of AG337 were determined, after the compound was shown to be stable in buffers of various pH, blank perfusate, and intestinal homogenate. The results indicated that absorption of AG337 was temperature-, pH-, Na+-, concentration-, and site-dependent. The best site of absorption is duodenum, where the absorption was 3-10 times (p < 0. 05) higher than absorption at jejunum, ileum, and colon. Among the four pH's studied, the best was at pH 6.5 (p < 0.05). Absorption was 80% lower in the absence of Na+, and 75% lower when the temperature of the perfusate was decreased to 4 degreesC. Permeability of AG337 also decreased about 75% when the concentration was raised to 100 microM. These results suggest that a nutrient carrier may be involved in the transport of AG337. To determine the carrier responsible for the absorption of AG337, its absorption was determined in the presence of various inhibitors at different concentrations. The results indicated that transport of AG337 was inhibited significantly (p < 0.01) by 100 microM of adenine, hypoxanthine, and xanthine. The transport was also inhibited significantly (p < 0.01) by a mixture of 100 microM each of adenine, hypoxanthine, and xanthine, but not by a mixture of 100 microM each of thymine and uracil. A higher concentration of hypoxanthine resulted in increased inhibition. In contrast, prototypical inhibitors of nucleoside transporter, dipyridamole and nitrobenzylthioinosine (NBMPR), did not significantly decrease the transport of AG337. The results also showed that absorption of AG337 had a significant nonsaturable component, with a nonsaturable Pw of 0.8. In conclusion, absorption of AG337 in the rat intestine has been shown to be mainly via a purine base carrier with a significant nonsaturable component.
Animals↗
Cell death attenuation by 'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex.
Apoptotic cell suicide initiated by ligation of CD95 (Fas/APO-1) occurs through recruitment, oligomerization and autocatalytic activation of the cysteine protease, caspase-8 (MACH, FLICE, Mch5). An endogenous mammalian regulator of this process, named Usurpin, has been identified (aliases for Usurpin include CASH, Casper, CLARP, FLAME-1, FLIP, I-FLICE and MRIT). This protein is ubiquitously expressed and exists as at least three isoforms arising by alternative mRNA splicing. The Usurpin gene is comprised of 13 exons and is clustered within approximately 200 Kb with the caspase-8 and -10 genes on human chromosome 2q33-34. The Usurpin polypeptide has features in common with pro-caspase-8 and -10, including tandem 'death effector domains' on the N-terminus of a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity, including the His and Cys which form the catalytic substrates diad, and residues that stabilize the P1 aspartic acid in substrates. Retro-mutation of these residues to functional caspase counterparts failed to restore proteolytic activity, indicating that other determinants also ensure the absence of catalytic potential. Usurpin heterodimerized with pro-caspase-8 in vitro and precluded pro-caspase-8 recruitment by the FADD/MORT1 adapter protein. Cell death induced by CD95 (Fas/APO-1) ligation was attenuated in cells transfected with Usurpin. In vivo, a Usurpin deficit was found in cardiac infarcts where TUNEL-positive myocytes and active caspase-3 expression were prominent following ischemia/reperfusion injury. In contrast, abundant Usurpin expression (and a caspase-3 deficit) occurred in surrounding unaffected cardiac tissue, suggesting reciprocal regulation of these pro- and anti-apoptotic molecules in vivo. Usurpin thus appears to be an endogenous modulator of apoptosis sensitivity in mammalian cells, including the susceptibility of cardiac myocytes to apoptotic death following ischemia/ reperfusion injury.
Alternative Splicing↗
Immunobiological studies on experimental visceral Leishmaniasis. V. The I-A(Bm12) mutation specifies resistance to infection.
The I-A(Bm12) mutation of the I-A beta subunit converted Leishmania donovani-susceptible C57BL/6 (B6) mice into the relatively resistant B6C-H-2 Bm12 (Bm12) strain. The relative resistance was reflected not only in the reduced splenic and hepatic parasite burden in Bm12 (compared with B6) but also by the ability of Bm12 mice to mount a T-cell proliferative response to parasite antigens. Assay of antileishmanial antibody (immunoglobulin G (IgG)2a and IgG1) in the sera of infected mice showed that in Bm12 mice the predominant isotype was IgG2a, rather than IgG1, whereas a similar level of both isotypes were found in B6 mice. From the serum immunoglobulin isotype titre it appeared that the antileishmanial T-cell response was biased towards a T helper (Th) 1 response in Bm12 mice whereas it was a mixed Th1 and Th2 response in B6 mice. These observations provide credence to the notion that polymorphism in class II major histocompatibility complex (MHC) molecules is responsible for the difference in the disease phenotype.
Animals↗
A fluorescence spectroscopic and molecular dynamics study of bis-ANS/protein interaction.
Despite emergence of bis-ANS as a major fluorescence probe of proteins structure, conformational and spectroscopic properties of protein/bis-ANS complexes remains largely unexplored. We have shown that fluorescence polarization of both ANS and bis-ANS is excitation wavelength dependent and this is a property of all protein-ANS/bis-ANS complexes studied. Bis-ANS excitation maximum is always more red shifted than the corresponding ANS complex. Even when corrected for the red shift, the bis-ANS complexes in some, but not all, cases show only a little lowering of polarization, suggesting modest additional depolarization in bis-ANS compared to ANS. Calculation of energy migration rate between the two rings suggests that energy migration rate should be high at all values of the naphthyl-naphthyl dihedral angle. Although, Molecular mechanics and dynamics calculations show that the lowest energy conformation of bis-ANS is when the two naphthalene rings are roughly perpendicular to each other, due to rapid energy migration this conformation should lead to dramatic lowering of emission anisotropy, unlike what is observed. Salt and temperature dependence of bis-ANS/protein interaction suggests little ionic interaction and pre-dominant interaction through hydrophobic aromatic rings. We conclude that bis-ANS binds to proteins through interaction with the aromatic rings and with two rings nearly parallel to each other.
Amino Acyl-tRNA Synthetases↗